The nematode C. elegans, discovered by Sidney Brenner in 1965, is the experimental organism (coliform bacillus, yeast, nematode, fly, South African clawed frog, zebrafish, mouse, etc.) that has been analyzed in the most detail in modem molecular biology. C. elegans is the simplest organism among experimental multicellular organisms. It also requires only approximately 3 days until a fertilized egg becomes an adult.
With multicellular organisms, an adult made up from many cells is basically produced by repeated sequential cell division of a single fertilized egg (a single cell). A dendrogram of a division sequence starting from a fertilized egg is referred to as “cell lineage”. C. elegans is the only multicellular organism for which cell lineage from a fertilized egg to adulthood has been clarified. This cell lineage was determined by Sulston et al. in 1983.
All normal (wild-type) C. elegans individuals exhibit identical cell lineage from egg fertilization to adulthood. In the case of mutation of specific genes, a change in the function of mutated genes causes a change in the pattern of cell division, i.e., cell lineage, from that of the wild type. A huge number of genes have been identified rapidly by an advance of research based on a presumption of the function of the mutated gene from the change of the cell lineage. The mass production of mutant animals has now begun. In consideration of the effective application of resources, automated analysis of cell lineage that is a starting point in the analysis of gene function is an essential technique.
For preparation of a conventional cell lineage, the so-called Nomarski microscope is used. In the Nomarski microscope, two beams of light (of identical wave form and phase, but having a very small difference in the light path) are generated by a set consisting of a polarizing plate and a Nomarski prism. The subject of observation is irradiated with these beams passing through the subject of observation. Differences in the refractive index of and optical path length through the sample produce different phases in the two beams of light after transmission. The two beams of transmitted light converge on the same optical path by the action of the set of polarizing plate and Nomarski prisms, but the phase difference between the two light beams causes interference. When using the Nomarski microscope, enhanced contrast produced by the action of interference facilitates observation. According to this method, the external shape and distribution of contents of a transparent subject is observed as contrasting light and dark areas. Biologically, a cell content and external shape (cell membrane), both transparent when using a common optical microscope, can be observed as areas of light and dark.
Sulston et al determined the cell lineage of C. elegans by preparing a sketch from images observed under a Nomarski microscope with the unaided eye. This consumed a considerable amount of time (probably 1 year or more) and labor.
More recently, cell lineage is generally prepared using a 4D microscopic image produced by employing the Nomarski microscope. A microscopic image yielded from observations made at specific focal points is regarded as a 2D (x−y axis) sectional image obtained by the action of cutting the subject of observation horizontally in a specific position. That is, moving the focal point up and down (moving along the z axis) yields a sectional image produced by cutting the subject of observation in various slices along the z axis. Unifying these images allows the reconstruction of the 3D shape of the subject of observation (3D image). Moreover, collection of a time series of 3D images allows temporal changes in the subject of observation to be followed. An image taken in this manner is referred to as a “4D (4-dimensional) microscopic image”.
Undoubtedly, present day methods for the construction of a 4D microscopic image are straightforward in comparison with those used at the time of Sulston's study. Nonetheless, considerable time and labor are still consumed, since decisions regarding the boundaries of the cell nucleus and cell membrane in the 4D image require input by the user. For example, a preparation from fertilized egg to 16-cells requires one day or more.
It is important to precisely recognize a nucleus area from the obtained image in order to construct the cell lineage. The recognition by image processing algorithms is incomplete, since in accordance with an increase in the number of cells, some areas (false positives) are recognized erroneously as the nuclear area. It is difficult to build up the cell lineage correctly from data containing many false positives. The operation for manually removing the misidentified nucleus areas from the result of the automatic recognition by human judgment is required.
The present invention sets out to make conventional preparation of the cell lineage more straightforward and has an object of providing a method for constructing cell lineage that is less labor intensive and requires less time as a result of using a computer.
Another object of the present invention is to efficiently remove misidentified nucleus areas (false positives).